The subject matter of the present application is related to that disclosed in each of the following U.S. patent applications which are being filed on the same day: U.S. Ser. Nos. 08/522,048; 08/522,434; 08/521,615; 08/522,435, the full disclosures of which are incorporated herein by reference.
1. Field of the Invention
The present invention relates generally to compositions and methods for detecting target substances in biological samples. More particularly, the present invention relates to labelling compositions comprising a multiplicity of fluorescent dye molecules bound to a plurality of binding molecules which in turn are bound to a linear polysaccharide backbone.
Many methods are known for detecting specific biological substances in solution and on solid phase supports. Of particular interest to the present invention are immunochemical methods where binding of a detectable label to a target substance in a biological sample is mediated by a specific binding substance. For example, a labelled specific binding substance, such as a fluorescently-labelled antibody, which recognizes the target substance (e.g., an antigen or hapten) is exposed to a biological specimen suspected of containing the target substance, and presence of the target substance is confirmed by binding of the label. Similar techniques may be performed using nucleic acid hybridization methods, where nucleic acid sequences of interest may be detected with a labelled polynucleotide which recognizes its complementary sequence in the nucleic acids of the biological specimen. In both cases, if present, the target substance will thus bind to the fluorescent label, allowing detection based on fluorescent emissions of the label.
Fluorescent labels offer a number of advantages compared with other common assay labels, such as enzyme labels. The detection of fluorescent labels is simple and does not require the addition of a substrate as is common with many enzyme labels. The signal generated by fluorescent label is localized, permitting the detection of multiple analytes in different reaction zones within a single reaction region or chamber. Thus, fluorescent labels allow simplified protocols for the detection of multiple analytes where only the final detection step must be performed separately for different analytes.
Of specific interest to the present invention, a class of cyanine fluorescent materials, referred to as "arylsulfonate cyanine fluorescent dyes," having particularly desirable properties has been developed. The arylsulfonate cyanine fluorescent dyes have high extinction coefficients (typically from 130,000 L/mole to 250,000 L/mole, good quantum yields, fluorescent emission spectra in a range (500 nm to 750 nm) which is outside of the autofluorescence wavelengths of most biological materials and plastics, good solubilities, and low non-specific binding characteristics. A particular arysulfonate cyanine fluorescent dye, designated Cy5, has the following structure: ##STR1##
Despite these excellent properties, arylsulfonate cyanine fluorescent dyes suffer from certain limitations. In particular, these dyes have a relatively narrow Stokes shift which results in significant overlap between the excitation and emission spectra of the dye. The overlap of excitation and emission spectra, in turn, can cause self-quenching of the fluorescence when the dye molecules are located close to each other when excited. Such self-quenching limits the number of arylsulfonate dye molecules which can be conjugated to a single antibody molecule for use in immunoassays. In the case of Cy5, an exemplary arylsulfonate cyanine fluorescent dye, the Stokes shift is 17 nm (which is the difference between an excitation wavelength of 650 nm and an emission wavelength of 667 nm). Optimal fluorescent yield is obtained when from two to four Cy5 molecules are conjugated to a single antibody molecule. The fluorescent signal output drops rapidly when more than four dye molecules are conjugated to a single antibody molecule. The inability to conjugate more than four dye molecules to individual antibody molecules significantly limits the sensitivity of immunoassays using Cy5-labelled antibodies and other binding substances.
The direct attachment of fluorescent dye molecules to carrier molecules, such as dextran, has been proposed as the technique for enhancing fluorescent output. Such carrier molecules would typically contain one or a few antibody molecules, where the binding of each antibody to a target substance will carry a number of fluorescent dye molecules. Attachment of arylsulfonate cyanine dyes to such carrier molecules, however, would not be expected to significantly increase the level of fluorescent emission because of the self-quenching density of the individual dye molecules, as described above. Moreover, the binding capacity of many prior fluorescent dyes/carrier molecules complexes has been limited, due both to a limited number of antibody molecules present in the complex and to steric hinderance of the antibodies from the carrier molecule.
For these reasons, it would be desirable to provide improved fluoresceht labelling compositions for use in the detection of target substances in biological samples. Such compositions should be useful in all circumstances where fluorescent labelling complexes generally find use, including solid phase immunoassays, immunohistochemical staining, flow cytometry, and the like. It would be particularly desirable to provide improved arylsulfonate cyanine dye compositions comprising conjugates having multiple dye molecules with reduced self-quenching and relatively high target-binding capacities. Such compositions should continue to display the low non-specific binding, high solubility, and other desirable characteristics associated with previous arylsulfonate cyanine dye formulations.
2. Description of the Background Art
Arylsulfonate cyanine fluorescent dyes are described in Mujumdar et al. (1993) BIOCONJUGATE CHEMISTRY 4:105-111; Southwick et al. (1990) CYTOMETRY 11:418-430; and U.S. Pat. No. 5,268,486. Cy5 is described in each of the references and is commercially available from Biological Detection Systems, Inc., Pittsburgh, Pa., under the tradename Fluorolink.TM. Cy5.TM.. Fluorescently labelled polysaccharides, such as dextrans, are described in Globe et al. (1983) ANAL. BIOCHEM. 130:287 and deBelder et al. (1973) CARBOHYDRATE RES. 30:376.